JPWO2018220670A1 - Observation device - Google Patents

Abstract

The observation device (1) according to the present invention emits illumination light upward from below the sample (X) for the purpose of observing a subject such as a cell without labeling without increasing the size of the device. An illumination optical system (6), and illumination light emitted from the illumination optical system (6) is reflected above the sample (X) and illuminates transmitted light transmitted through the sample (X) below the sample (X). An objective optical system (5) for taking an image on a different path from the optical system (6), wherein the objective optical system (5) partially reduces the transmittance near the pupil plane and modulates the phase of light. An illumination optical system including a modulation element, an illumination optical system configured to restrict a light from the light source to a specific emission area, and an illumination area restriction unit; The emission area (6e1) by (6c1) is set in the direction orthogonal to the optical axis (A) of the objective optical system (5). And a section capable of illuminating position changing mechanism (6c2).

Description

  The present invention relates to an observation device.

  As an apparatus for observing a subject such as a cell without labeling, an observation apparatus using a phase difference observation method or a differential interference observation method is known (for example, see Patent Document 1).

JP-A-7-261089

However, the observation apparatus disclosed in Patent Document 1 needs to dispose a photographing optical system and an illumination optical system with a subject interposed therebetween, and thus has a disadvantage that the apparatus becomes large and complicated.
The present invention has been made in view of the above-described circumstances, and has as its object to provide an observation apparatus that can observe a subject such as a cell without labeling without increasing the size of the apparatus.

  One embodiment of the present invention is an illumination optical system that emits illumination light upward from below the sample, and the illumination light emitted from the illumination optical system is reflected above the sample and transmitted through the sample. An objective optical system for photographing transmitted light under a path different from the illumination optical system below the sample, wherein the objective optical system partially reduces the transmittance near the pupil plane and modulates the phase of light. The illumination optical system includes a light source, an illumination area restriction unit that restricts light from the light source to a specific emission area, and an emission area defined by the illumination area restriction unit. An observation device including an illumination position variable mechanism that can be adjusted in a direction perpendicular to the optical axis.

  According to this aspect, after the illumination light emitted from the light source is emitted upward from below the sample, it is reflected above the sample and transmitted through the sample from above. The transmitted light transmitted through the sample is photographed by an objective optical system that is different from the illumination optical system disposed below the sample. Since both the light source and the objective optical system are arranged below the sample, the subject such as cells can be observed without labeling by photographing the transmitted light without increasing the size of the apparatus.

  The light emitted from the light source is illuminated on the sample as illumination light whose emission area is limited by the illumination area limiting unit, and is incident near the pupil plane of the objective optical system. The pupil modulation element arranged near the pupil plane has a region where the transmittance is partially reduced, so that light not passing through the sample passes through the region where the transmittance is partially reduced. By setting, light that does not pass through the sample is attenuated and reaches the image plane with a further delay in phase.

  On the other hand, light that has passed through the sample is divided into light that is diffracted by the microstructure in the sample and light that does not diffract (0th-order diffracted light). The zero-order diffracted light is attenuated by passing through a region where the transmittance of the pupil modulation element arranged near the pupil plane is partially reduced, and reaches the image plane with a further delay in phase. The light diffracted by the sample passes through the region of the pupil modulation element where the transmittance is not reduced, so that it does not attenuate much and reaches the image plane with a phase delayed by π / 4.

  As a result, light that has not passed through the sample is attenuated by the pupil modulator, and becomes darker. Since the light that has passed through the sample has the same phase delay amount for both the zero-order diffracted light and the diffracted light, the phase difference between the two becomes zero. , Interfere and form an image that is brighter than the light that did not pass through the sample. That is, when the sample is a cell, the cell is isotropic without brightness unevenness, and a bright image observable even to a fine structure in the cell can be obtained by using the diffracted light.

  In this case, according to this aspect, even if the reflection surface disposed above the sample is inclined, the operation of the illumination position variable mechanism causes the emission area by the illumination area restriction unit to move along the optical axis of the objective optical system. By adjusting in the direction orthogonal to the above, the emission area of the illumination light can be adjusted to satisfy the above condition.

In the above aspect, the illumination position variable mechanism may be capable of individually adjusting the emission regions arranged at a plurality of positions spaced apart in a circumferential direction around the optical axis in a direction orthogonal to the optical axis. .
By doing so, it is possible to perform observation with a bright image in which the sample is distinguished by illumination light from a plurality of directions.

Further, in the above aspect, the illumination area restriction unit may be a mask, and the illumination position variable mechanism may move the mask.
This makes it possible to easily adjust the emission area of the illumination light.

Further, in the above aspect, the illumination region limiting section has a flat fixed mask having a plurality of windows through which the illumination light passes in a direction along the optical axis, and the fixed mask is formed with respect to the fixed mask. A plurality of movable masks that can be moved in different directions along the surface.
In this way, only the illumination light passing through the plurality of windows is limited by the fixed mask, and by moving the movable mask with respect to the fixed mask, a part of the illumination light limited by the fixed mask is Can be easily adjusted.

Further, in the above aspect, the movable mask includes a plurality of windows that are rotatably supported around the optical axis and are arranged at different circumferential positions around the optical axis at different radial positions. You may.
With this configuration, by rotating the movable mask around the optical axis, the window for emitting the illumination light can be switched, and the emission area can be moved in the radial direction.

Further, in the above aspect, the light source includes a plurality of light source units, and the illumination region limiting unit emits the illumination light from each of the light source units from a different emission region in a direction orthogonal to the optical axis. The lighting position variable mechanism may alternatively light the light source unit.
In this manner, by switching the light source unit to light up, it is possible to easily switch the emission region from which the illumination light is emitted.

Further, in the above aspect, the illumination region restriction unit may include an optical connection mechanism that guides the illumination light from each of the light source units to each of the emission regions while approaching the illumination light.
In order to converge the illumination light from the light source with a collimating lens and accurately match the projected image of the illumination light with the pupil plane of the objective optical system, it is necessary to secure a sufficiently large focal length of the collimating lens. Although the height dimension from the light source to the emission area becomes large, by providing the optical connection mechanism, the emission area can be brought close even if each light source is large, and the height dimension can be reduced to achieve a compact configuration. .

Further, in the above aspect, the optical connection mechanism may partition the illumination light from each of the light sources so as not to mix.
With this configuration, when the light source is switched, the illumination light can be emitted only from the emission region corresponding to the light source.

Further, in the above aspect, the optical connection mechanism may be a light guide fiber.
By doing so, the illumination light emitted from the light source is guided by the light guide fiber, and can be emitted from the emission region having a smaller area than the light source. By bending the light guide fiber, it is possible to reduce the height dimension and make the light guide fiber compact.

  ADVANTAGE OF THE INVENTION According to this invention, there exists an effect that a subject, such as a cell, can be observed without labeling, without enlarging an apparatus.

It is a longitudinal section showing an observation device concerning a 1st embodiment of the present invention. FIG. 2 is a plan view illustrating an example of an arrangement of light sources provided in the observation device in FIG. 1. FIG. 2 is a plan view showing a fixed mask provided in the observation device in FIG. 1. FIG. 2 is a plan view illustrating an example of a movable mask provided in the observation device in FIG. 1. FIG. 2 is a plan view illustrating an example of a pupil modulation element provided in the observation device in FIG. 1. FIG. 2 is a longitudinal sectional view of an objective optical system illustrating an operation of the observation device in FIG. 1. FIG. 2 is a diagram illustrating an example of an image acquired by the observation device in FIG. 1. FIG. 2 is a plan view illustrating an example of a projected image of an opening of a movable mask in a pupil modulation element when a top plate of a container is tilted in the observation device in FIG. 1. FIG. 2 is a diagram illustrating a change in an average value of brightness of an image acquired by an imaging device when a movable mask is moved in the observation device in FIG. 1. FIG. 5 is a longitudinal sectional view of an illumination optical system showing a first modification of the observation device in FIG. 1. FIG. 11 is a plan view illustrating an example of a fixed mask provided in the illumination optical system of FIG. 10. FIG. 11 is a plan view illustrating an example of a movable mask provided in the illumination optical system of FIG. 10. FIG. 13 is a plan view showing a state in which the movable mask of FIG. 12 is rotated with respect to the fixed mask of FIG. 11 and one of the openings is aligned. It is a longitudinal cross-sectional view which shows the 2nd modification of the observation device of FIG. It is a longitudinal cross-sectional view which shows the 3rd modification of the observation device of FIG. It is a longitudinal cross-sectional view which shows the 4th modification of the observation device of FIG.

An observation apparatus 1 according to a first embodiment of the present invention will be described below with reference to the drawings.
As shown in FIG. 1, an observation device 1 according to the present embodiment includes a stage 3 on which a container 2 containing a sample X such as a cell is placed, and is disposed below the stage 3, and the stage 3 is moved from above. An objective lens 5a for condensing the transmitted light; an objective optical system 5 for photographing the light transmitted through the sample X; and an objective lens 5a disposed radially outward of the objective optical system 5 and transmitted through the stage 3 and upward. An illumination optical system 6 having a different path from the objective optical system 5 that emits illumination light, and a control unit (not shown) for controlling the illumination optical system 6 are provided.

The stage 3 includes a mounting table 3a made of an optically transparent material, for example, glass so as to cover the upper part of the objective optical system 5 and the illumination optical system 6, and the container 2 is mounted on the upper surface of the mounting table 3a. It has become so.
The container 2 is, for example, a cell culture flask having a top plate 2a, and is entirely made of an optically transparent resin.

  As shown in FIG. 2, the illumination optical system 6 includes four LED light sources (light sources) 6a arranged at equal intervals in the circumferential direction outside the objective optical system 5, and light from each of the LED light sources 6a. Plate 6b for diffusing light, a mask 6c provided close to the diffuser plate 6b, and restricting illumination light from the LED light source 6a to a specific emission region, and illumination for gradually diffusing from the restricted emission region A collimating lens 6d for converting light into substantially parallel light.

  The mask 6c includes a flat fixed mask (illumination area limiter) 6c1 and a flat movable mask (illumination position variable mechanism) 6c2 that are arranged on the upper surface of the diffusion plate 6b in the thickness direction. As shown in FIG. 3, the fixed mask 6c1 has fan-shaped openings (emission areas, window portions) 6e1 for transmitting four illumination lights at equal intervals in the circumferential direction in a ring-shaped light-shielding member. Have.

  Further, as shown in FIG. 4, the movable mask 6c2 is disposed in a position to close each opening 6e1 of the fixed mask 6c1, and has a fan-shaped light shielding member whose radial dimension is smaller than the opening 6e1 of the fixed mask 6c1. An opening (emission area, window) 6e2 is provided. Each movable mask 6c2 is supported movably in the radial direction of the fixed mask 6c1, and the position of the opening 6e2 in the radial direction is adjusted by the control unit. Thereby, the position of the opening 6e2 from which the light from the LED light source 6a is emitted can be adjusted in the radial direction.

In the present embodiment, the collimator lenses 6d are arranged above the opening 6e2 and at four places in the circumferential direction around the optical axis A of the objective optical system 5. Preferably, the respective collimating lenses 6d are arranged at equal intervals in the circumferential direction on a circle centered on the optical axis A.
Further, in the circumferential direction of the optical axis A, the optical axis B of the collimating lens 6d is disposed closer to the optical axis A of the objective optical system 5 than the position of the opening 6e2. Thus, the light incident from the LED light source 6a is made substantially parallel light, and the parallel light flux is emitted while being inclined in the direction along the optical axis A.

Further, as shown in FIG. 14, a single collimating lens 6d provided with a through hole 6f in the center and the objective optical system 5 may be arranged in the through hole 6f.
The substantially parallel light that is emitted while being inclined by the collimating lens 6d is reflected by the top plate 2a of the container 2 arranged above, and the liquid Y, the sample X, and the mounting table 3a of the stage 3 in the container 2 below. After passing through the objective optical system 5.

  As shown in FIG. 6, the objective optical system 5 includes an objective lens 5a for condensing transmitted light incident from above, a pupil modulation element 5b disposed near a pupil plane, a flare stop 5g, and an imaging lens 5c. And an image sensor 5d. As shown in FIG. 5, the pupil modulation element 5b has a ring-shaped phase film inside a brightness stop 5e arranged near the pupil plane, at a position radially away from the center of the pupil plane by a predetermined distance. 5h.

  The phase film 5h has a function of partially lowering the transmittance and modulating the phase of the transmitted light to delay by π / 4. Reference numeral 5f denotes a glass plate that supports the aperture stop 5e and the pupil modulation element 5b.

The operation of the observation device 1 according to the present embodiment thus configured will be described below.
As shown in FIG. 1, the illumination light emitted from the LED light source 6a of the illumination optical system 6 passes through the opening 6e2 of the movable mask 6c2, and as a light flux restricted to an emission area having a predetermined size. The light beam is emitted upward, passes through the collimating lens 6d disposed above, is converted into substantially parallel light, and becomes a light beam inclined toward the optical axis A of the collimating lens 6d.

  The substantially parallel light traveling upward from the collimating lens 6d passes through the mounting table 3a constituting the stage 3, the bottom surface of the container 2, and the liquid Y, is reflected by the top plate 2a of the container 2, and is obliquely downward. Oblique illumination is applied from above. Then, the transmitted light transmitted through the container 2 is condensed by the objective lens 5a after passing through the bottom surface of the container 2 and the mounting table 3a, passes through the pupil modulation element 5b, and is imaged by the imaging lens 5c. Photographed by 5d.

  That is, the illumination light, which is substantially parallel light transmitted through the sample X from obliquely above, is transmitted through the sample X and collected by the objective lens 5a. As shown in FIG. 6, the transmitted light that has passed through the region where the sample X does not exist is incident on the objective lens 5a without being refracted and remains substantially parallel light, so that it is arranged on the pupil plane of the objective lens 5a. After the images of the openings 6e1 and 6e2 of the mask 6c are projected on the pupil modulation element 5b, the image is formed by the imaging lens 5c and is imaged by the imaging element 5d.

  The transmitted light transmitted through the region where the sample X exists is refracted by the fact that the refractive index of the sample X is different from the surrounding refractive index. In FIG. 6, the light rays a and e that do not pass through the sample X and the light ray c that is orthogonally incident on the surface of the sample X pass through the phase film 5h of the pupil modulation element 5b without being refracted, and are reduced by the phase film 5h. Construct an image of the brightness.

  On the other hand, in FIGS. 6A and 6B, light beams b and d transmitted through the sample X are diffracted light (indicated by broken lines in the figure) and non-diffracted light (0th-order diffracted light) that are transmitted through the sample X and diffracted by a minute structure in the sample X. ). The zero-order diffracted light passes through the phase film 5h of the pupil modulation element 5b, is attenuated, and reaches the image plane with a further delay in phase. Since the diffracted light in the sample X passes through the outside of the phase film 5h, it is not attenuated, and reaches the image plane at the same position as the 0th-order diffracted light with a phase delayed by π / 4.

  As a result, the light that has not passed through the sample X is attenuated by the pupil modulator 5b and becomes dark, and the light that has passed through the sample X has the same phase delay amount for both the 0th-order diffracted light and the diffracted light, so that the phase difference Becomes 0 and interferes to form an image brighter than the light that has not passed through the sample X. That is, when the sample X is a cell, the cell is isotropic without brightness unevenness, and as shown in FIG. Can be obtained.

  In this case, if the top plate 2a of the container 2 placed on the stage 3 is inclined or curved for some reason, the light is emitted from the emission area, reflected on the top plate 2a, and passes through the sample X. And the light condensed by the objective lens 5a does not pass through the phase film 5h. For example, as shown in FIG. 8, a projection image (indicated by hatching in the figure) of the exit area on the pupil modulation element 5b is deviated from the phase film 5h. In such a case, since the above-described interference does not occur, an image in which the sample X stands out from other parts cannot be obtained.

  In such a case, according to the present embodiment, by moving the movable mask 6c2 in the radial direction with respect to the fixed mask 6c1, the emission area is moved in the radial direction, and the reflected light is reflected on the top plate 2a and the objective lens The position can be adjusted so that the light condensed by 5a passes through the phase film 5h.

More specifically, when the movable mask 6c2 is moved in the radial direction, the average value of the brightness of the image acquired by the image sensor 5d changes as shown in FIG.
That is,
(1) When the opening 6e2 of the movable mask 6c2 is close to the optical axis A, it is cut off by the frame of the objective optical system 5 or the like, so that the average brightness of the image is low.
(2) When the blur is reduced, the average value of the brightness of the image increases,
(3) When no blurring occurs, the average brightness of the image becomes constant,
(4) When the projected image coincides with the phase film 5h, the average value of the image brightness temporarily decreases.
(5) When the projected image deviates from the phase film 5h, the average value of the brightness of the image becomes constant again,
(6) When the aperture 6e2 of the movable mask 6c2 moves away from the optical axis A, the aperture is cut by the aperture stop 5e, and the average value of the image brightness decreases.

Therefore, by detecting the position (4) by moving the movable mask 6c2 in the radial direction, the projected image of the emission area can be made to coincide with the phase film 5h. By performing this operation for each of the four movable masks 6c2, the projected image of the emission area can be arranged at a position overlapping the phase film 5h in all directions.
That is, according to the observation apparatus 1 according to the present embodiment, even if the top plate 2a of the container 2 mounted on the stage 3 is tilted or deformed for some reason, the sample X stands out from other parts. There is an advantage that observation with a high-contrast image can be performed.

Note that, in the present embodiment, only the movable mask 6c2 is moved, but instead, the LED light source 6a and the mask 6c may be moved integrally. Thereby, the light from the LED light source 6a can be used efficiently.
Further, in the present embodiment, the position of the opening 6e2 of the movable mask 6c2 in the radial direction is adjusted by the control unit, but instead, a manually adjusted one may be adopted.

  In the present embodiment, the opening 6e2 is moved in the radial direction by moving the movable mask 6c2 in the radial direction with respect to the fixed mask 6c1, but instead of this, FIGS. As shown, the opening 6e2 may be switched in the radial direction by rotating the movable mask 6c2 around the optical axis A.

  That is, as shown in FIG. 11, the fixed mask 6c1 is provided with a plurality of fan-shaped openings 6e1 at equal intervals in the circumferential direction and spaced apart in the radial direction, and the movable mask 6c2 has As shown in FIG. 12, openings 6e2 are provided at positions different in the circumferential direction and the radial direction by the same number as the openings 6e1 of the fixed mask 6c1. As shown in FIG. 10, four movable masks 6c2 are arranged in a stacked state, and are individually rotatably provided corresponding to the openings 6e1 at respective positions in the circumferential direction of the fixed mask 6c1. That is, four movable masks 6c2 having the form shown in FIG. 12 are stacked with the positions of the openings 6e2 being different by approximately 90 °.

  By rotating the movable mask 6c2 corresponding to the opening 6e1 at each position in the circumferential direction of the fixed mask 6c1, as shown in FIG. 13, one of the openings 6e2 of the movable mask 6c2 can be selectively used as the fixed mask 6c1. The opening can be made to coincide with any one of the openings 6e1, whereby the emission region can be moved in the radial direction as described above. FIG. 13 shows an example of the positional relationship between the fixed mask 6c1 arranged in one direction in the circumferential direction of the fixed mask 6c1 and the corresponding movable mask 6c2.

  In the above-described embodiment, the emission area is switched by moving the movable mask 6c2. Alternatively, as shown in FIG. 14, a plurality of LED light sources (light source units) 6a arranged in the radial direction may be used. May be arranged corresponding to each opening 6e1 of the fixed mask 6c1 as in FIG. 10, and the emission area may be moved in the radial direction by switching the LED light source 6a to be turned on. A light-shielding wall 6g is provided between the openings 6e1 of the fixed mask 6c1 so as to block light from the adjacent LED light source 6a. In this case, the lighting of the LED light source 6a is controlled by the control unit (illumination position variable mechanism).

  As shown in FIG. 15, the LED light sources 6a are arranged obliquely so that the optical axes of the LED light sources 6a arranged in the radial direction are close to each other, and a collimating lens 6h is attached to each LED light source 6a. Alternatively, a light guide path (illumination area limiter, optical connection mechanism) 6i that separates the light from each LED light source 6a may be provided between the diffusion plate 6b and the collimator lens 6h. In the example shown in FIG. 15, the light guide path 6i has a cross-sectional shape that gradually narrows from the collimating lens 6h toward the diffusion plate 6b. Can be limited to small. Thereby, the focal length of the collimator lens 6h can be shortened, the distance from the collimator lens 6h to the diffusion plate 6b can be shortened, and the height of the illumination optical system 6 can be suppressed.

  In this case, the inner surface of the light guide path 6i may be constituted by a mirror surface or may be filled with a glass rod. Further, a lens having a negative power may be disposed above the diffusion plate 6b to increase the diffusion angle.

  Further, instead of the hard light guide path 6i, a light guide fiber bundle (illumination area limiting part) 6k may be adopted as shown in FIG. In this case, the emitted light is expanded by the exit numerical aperture of each light guide fiber (optical connection mechanism) 6k1 constituting the light guide fiber bundle 6k, so that the diffusion plate 6b may not be provided. In this case, the degree of freedom in the arrangement of the LED light source 6a serving as a light source can be improved, the LED light source 6a can be disposed at a relatively distant position, and the temperature rise of the sample X can be prevented.

Further, in each of the above embodiments, the emission area of the illumination light is set at four places in the circumferential direction around the optical axis A of the objective optical system 5, but instead, an arbitrary number of one or more places may be provided. You may.
Although the phase film 5h is arranged in an annular shape, a plurality of fan-shaped phase films 5h spaced in the circumferential direction may be arranged in accordance with the number of emission regions.

  Although the phase of the light is delayed by π / 4 in the phase film 5h, the phase may be delayed by 3π / 4 instead. As a result, the phase delay of the diffracted light with respect to the 0th-order diffracted light can be set to π, and the image of the sample X is made darker than its surroundings due to the interference between the 0th-order diffracted light and the diffracted light, thereby obtaining a distinct image. Can be.

Reference Signs List 1 observation apparatus 5 objective optical system 5b pupil modulation element 6 illumination optical system 6a LED light source (light source, light source unit)
6c1 Fixed mask (Illumination area limiting part)
6c2 Movable mask (variable illumination position mechanism)
6e1 opening (injection area, window)
6e2 opening (injection area, window)
6i Light guide path (illumination area limiter, optical connection mechanism)
6k light guide fiber bundle (illumination area limiter)
6k1 light guide fiber (optical connection mechanism)
A Optical axis X Sample

Claims (9)

An illumination optical system that emits illumination light upward from below the sample,
An objective optical system configured to photograph the transmitted light transmitted through the sample by reflecting the illumination light emitted from the illumination optical system above the sample and below the sample by a different path from the illumination optical system. ,
The objective optical system includes a pupil modulation element that partially reduces transmittance and modulates the phase of light in the vicinity of the pupil plane,
The illumination optical system, a light source, an illumination area limiting unit that limits light from the light source to a specific emission area, and the emission area by the illumination area limitation unit in a direction orthogonal to the optical axis of the objective optical system. An observation device comprising an adjustable illumination position variable mechanism.   The observation device according to claim 1, wherein the illumination position variable mechanism is capable of individually adjusting the emission regions arranged at a plurality of positions spaced apart in a circumferential direction around the optical axis in a direction orthogonal to the optical axis. . The illumination area limiting unit is a mask,
The observation device according to claim 1, wherein the illumination position variable mechanism moves the mask.   The illumination region restricting section has a flat fixed mask having a plurality of windows through which the illumination light passes in a direction along the optical axis, and a different direction along the surface of the fixed mask with respect to the fixed mask. The observation apparatus according to claim 3, further comprising a plurality of movable masks that are moved.   The observation apparatus according to claim 4, wherein the movable mask is rotatably supported around the optical axis, and includes a plurality of windows arranged at different circumferential positions around the optical axis at different radial positions. . The light source includes a plurality of light source units,
The illumination region restriction unit restricts the illumination light from each of the light source units to be emitted from a different one of the emission regions in a direction orthogonal to the optical axis,
The observation device according to claim 1, wherein the illumination position variable mechanism selectively turns on the light source unit.   The observation device according to claim 6, wherein the illumination region restriction unit includes an optical connection mechanism that guides the illumination light from each of the light source units to each of the emission regions while approaching the illumination light.   The observation device according to claim 7, wherein the optical connection mechanism partitions the illumination light from each of the light sources so as not to mix.   9. The observation device according to claim 7, wherein the optical connection mechanism is a light guide fiber.

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